Surfactant, a material highly enriched with phosphatidylcholine (PC), is deficient in asthma. Tumor necrosis factor alpha (TNFa) plays a key role in asthma and decreases surfactant PC synthesis. The major question addressed in this proposal is how TNFa decreases PC synthesis during airway inflammation? Prior studies in the PI's laboratory have shown that the bioactive sphingolipid, sphingosine generated in response to TNFa activation of the sphingomyelin hydrolysis pathway, is one important mechanism whereby TNFa exerts its inhibitory effects on surfactant PC synthesis. This proposal will expand on these observations by investigating the molecular basis by which TNFa-sphingosine signaling inhibits surfactant PC synthesis. The synthesis of PC in cells is tightly regulated by the rate-limiting enzyme cytidylyltransferase (CT). CT activity is inhibited by sphingosine and by enzyme phosphorylation. The physiologic role of CT phosphorylation and regulation of CT gene transcription, however, remain largely unknown. One effect of TNFa is the activation of multiple kinase pathways, including the c-Jun N-terminal kinase (JNK) pathway. Preliminary data by the PI demonstrate that i) sphingosine and JNK levels are elevated but CT expression decreased in a murine ovalbumin model of airway inflammation, and ii) sphingosine coordinately activates JNK, increases CT phosphorylation, and decreases CT mRNA. Thus, TNFa activation of JNK leading to CT phosphorylation or CT gene transcriptional repression by sphingosine might represent a novel effector mechanism for the inhibitory effects of TNFa on surfactant PC synthesis in allergic airway inflammation. We will specifically test the hypothesis that TNFa inhibits surfactant PC synthesis by coordinately increasing CT phosphorylation (AIM 1) and decreasing CT gene transcription (AIM 2) thereby reducing surfactant availability in a murine model of allergic inflammation. We will determine if negative effects of TNFa on CT activity are due to sphingosine activation of JNK (AIM 1). We will also determine if sphingosine inhibits CT gene transcription (AIM 2). In Aim 1 our hypothesis will be tested using molecular and biochemical approaches to identify the regions within the CT primary structure that are targets for site-specific phosphorylation by JNK. We will perform site-directed mutagenesis of CT to generate enzyme mutants, that when expressed in vivo, are less sensitive to JNK-induced phosphorylation. In Aim 2, we will perform deletional and mutational analysis of the CT promoter to localize sphingosine-regulated c/s-acting negative regulatory element(s) (NRE). Finally, a portion of the 5' flanking region of the CT gene including these elements was coupled to a reporter gene (b-galactosidase) and used to generate transgenic promoter-reporter mice. We will test the hypothesis that these elements are sufficient to confer TNFa-repression of CT gene transcription within a model of allergic (Th2) inflammation. Our hypothesis will be tested using an ovalbumin murine model of airway inflammation with analysis conducted in primary type II alveolar epithelial cells. These studies will be supplemented with a TNFa-responsive murine type II cell line. The unique contributions of this proposal impacting the field of asthma include 1) delineation of a novel kinase pathway linking TNFa-signaling with surfactant synthesis, 2) studies investigating CT gene transcriptional repression which represent a relatively new regulatory mechanism for this key surfactant enzyme, and 3) for the first time, studies directed at stimulating surfactant synthesis by expression of novel CT mutants that exhibit robust catalytic activity and are phosphorylation-resistant in the setting of cytokine-associated airway inflammation.